The mechanistic target of rapamycin (mTOR) responds to diverse environmental signals to control essential cellular functions, playing critical roles in processes related to metabolism, tumorigenesis, and immune function. mTOR constitutes the catalytic core of two functionally distinct signaling complexes, mTORC1 and mTORC2: mTORC1 promotes anabolic cellular processes; while significantly less well understood; mTORC2 promotes cell survival and modulates the actin cytoskeleton. Despite the clear physiologic and therapeutic importance of mTOR, fundamental gaps exist in our knowledge regarding cellular mTOR regulation, especially with regard to the molecular pathways that regulate mTOR activity. Exciting recent work from our laboratory revealed that mTOR phosphorylation plays an important and previously unrecognized role in mTORC1 function. Using phospho specific antibodies and an in vitro kinome screen as tools, we discovered that TBK1/IKKe (kinases that promote innate immune signaling and the host defense response) and AMPK (a kinase that responds to energetic stress) phosphorylate mTOR on distinct sites. Importantly, these phosphorylation events occur in response to physiological signals in cultured cells and in vivo (i.e. mice). Our preliminary data indicate that TBK1/IKKe-mediated phosphorylation of mTOR S2159 promotes mTORC1 signaling while AMPK promotes mTORC2 signaling in response to acute energetic stress. In this application we propose to elucidate roles for these physiologically and pathologically significant signaling molecules in the cellular regulation and function of mTOR. In Aim 1, we investigate the hypothesis that TBK1 and IKKe act directly on mTORC1 to promote growth factor responses and innate immunity; in Aim 2, we investigate the hypothesis that AMPK acts directly on mTORC2 to suppress apoptosis and enhance mitochondrial function to maintain energy homeostasis during acute energetic stress. Emerging data suggest that TBK1/IKKe contribute to chronic inflammatory diseases and obesity-linked insulin resistance; moreover, cancer cells hijack TBK1/IKKe to promote tumorigenesis. As cellular TBK1/IKKe signaling networks remain poorly understood, this research focused on the TBK1/IKKe-mTORC1 axis has potential to impact the future clinical management of chronic inflammatory diseases and cancer. In response to insufficient ATP levels, AMPK stimulates energy producing catabolic and suppresses energy consuming anabolic pathways. This research focused on the AMPK-mTORC2 axis may explain in part the emerging paradox that AMPK can act as a tumor suppressor (by inhibiting cellular anabolism) and a tumor promoter (by inhibiting apoptosis), depending on cellular context. This work will unveil novel regulatory paradigms that directly impact mTOR and will reveal previously unknown links between mTOR and the processes controlled by these kinases, including cell survival, cell metabolism, tumorigenesis, and innate immunity. As the signals that regulate mTORC2 remain virtually unknown, the identification of AMPK as an mTORC2 activator represents a particularly important milestone.